Field of the Invention
The present invention relates to devices for processing biological samples. In particular, the invention concerns a microtiter plate assembly comprising microtiter plates having a plurality of sample wells. Such plates are used, for example, in thermal cyclers for performing a Polymerase Chain Reaction (abbreviated “PCR”) process. The present invention also concerns a method of processing biological samples.
Description of Related Art
Biological samples are processed in industrial and clinical diagnostics, pharmaceutical and research applications, and as processes have improved, the need for increasing the number and speed of samples processed has also increased. This has led to a standardization of sample containers based on a few, historically significant standards that have allowed users to utilize a large number of instruments or robotic handlers which were designed to operate within that standard.
The standards that are most commonly used are based on the formats of the microfuge tube, the microscope slide and the microtiter plate. Microfuge tubes come in several, usually non-interchangeable, sizes based on the desired size of the sample to be processed, and are usually used for liquid samples volumes of more than 0.01 ml to 1.5 ml. Microscope slides are utilized for tissue samples and very high density arrays of tiny samples that can be bound to the surface of the slide. Microtiter plates are built like arrays of very small microfuge tubes, and are available in a multitude of formats with varying materials, well geometries and sample densities, but all share the same basic footprint and are typically used for liquid samples that are between 10 μl and 500 μl in volume. It is interesting to note that several new technologies that have seen intensive development efforts in recent years, including microarrays and microfluidics applications, are almost always implemented to conform to one of these three standards in order to take advantage of the many tools developed for these standards.
Whilst the microfuge tube offers relatively high volume of reaction and low throughput of biological samples, the trend for clinical diagnostics, industrial microbial detection, and pharmaceutical and academic research has been able to reduce the volume of reaction and increase the throughput of these processes. To this end, high density microtiter plates and slide-based microarrays have become more commonly used. These formats are of particular interest because they offer the ability to perform parallel experiments, reduce reagent consumption, and confer the potential to utilize smaller, relatively less expensive laboratory and analytical instrumentation.
Microtiter plates are approximately 85 mm wide by 127 mm long by as many as 25 mm high. They come in several formats, but for molecular biology applications, 96-well and 384-well formats are, by far, the most common. 96-well microtiter plates typically consist of an 8×12 array of conical-shaped wells of 9 mm center-to-center pitch and an inner diameter of 5 to 6 mm. Depending on the variety of plate, each well can hold a maximum of 100 μl to 200 μl of reaction volume. 384-well plates halve the spacing, such that the plates now offer a 16×32 format, with 4.5 mm pitch, 3 to 3.5 mm inner diameters, and maximum sample volumes of 40 μl to 50 μl. Most biological chemistries performed in a microtiter plate are solution-based, but surfaced based chemistries can also be performed.
Microslide-sized arrays come in a variety of sample densities, but have the following general aspects in common: i) footprint of microarray typically is 25 mm by 75 mm, ii) generally based upon surface chemistries, and iii) typically do not have an individual three-dimensional aspect for addressing each sample. Sample densities can vary from a few thousand to over a hundred thousand per slide.
Microtiter plates and microarrays differ in their ability to address individual samples. Microtiter plates offer the well-to-well spacing and 3-dimensional aspect of a sample vessel, so that each well can be manipulated individually allowing for variation of both sample and reactants across a single plate. On the other hand, microscope-sized microarrays, for the most part, do not allow for every permutation of sample and reactants to be performed on a single microarray slide. The reason for this key difference is that the spacing on microtiter plates allows for standard pipetors and liquid handling robotic stations to both add and remove liquid from each well—thus allowing for unique combinations of sample and reactants to be applied across a single plate. Microarrays, however, tend neither to have the 3-D aspect to sample containment, nor the ability to be addressed by standard pipetors and liquid handling robots, which are generally required for such individualized reaction manipulation. It should be noted that there are a handful of microscope slide-based vessels that contain thousands to hundreds of thousands of pits or holes that confer a three-dimensional space in which to perform liquid-phase reactions. However, because the volumes of such spaces are measured in the picoliter range, and the density is so great, individual sample manipulation is impossible with commercially available liquid handler devices.
Thermal cyclers are instruments commonly used in molecular biology for applications such as PCR and cycle sequencing, and a wide range of instruments are commercially available. A subset of these instruments, which include built-in capabilities for optical detection of the amplification of DNA, are referred to as “real-time” instruments. Although these can sometimes be used for different applications than non-real-time thermal cyclers, they operate under the same thermal and sample preparation parameters.
The important parameters that govern how well a thermal cycler operates are: uniformity, accuracy and repeatability of thermal control for all the samples processed, ability to operate in the environment of choice, speed of operation, and sample throughput. As the processes get more complicated and the amount of automation increases, the importance of compatibility with and flexibility between different process phases and technologies is emphasized.
Sample throughput needs have come about over time. All currently produced thermal cyclers can be divided up into groupings based on how they accommodate samples. The first instruments were built to accommodate a small number of tubes which were individually processed and loaded into the cycler (example: Perkin-Elmer 4800). As sample throughput needs grew, instruments were developed to accommodate plastic trays (microtiter plates) that were essentially arrays of 96 or 384 tubes (examples: Perkin-Elmer 9600, MJ Research PTC-200, Eppendorf MASTERCYCLER®). Both of these formats tend to utilize metal blocks to heat and cool the tubes, which places some limits on the speed of thermal cycling due to the time needed to heat and cool the mass of the metal block.
The vast majority of thermal cyclers in use today are block based thermal cyclers that accommodate microtiter plates. The reason for this, despite the potential for slow cycling speeds of these instruments, is that microtiter plates can be used with a wide range of liquid volumes, and the actual sample throughput is tends to be quite high in terms of total number of samples that can be processed in a given timeframe. This last aspect is only partially a function of the instrument itself; it is also dependent upon the equipment that is available to process and load the samples both before and after the thermal cycling reaction. The vast majority of microtiter plates in use conform to a set of standards codified by the Society for Biomolecular Screening (SBS) over the last decade. The plates typically have 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. The standard also governs well dimensions (e.g. diameter, spacing and depth) as well as plate properties (e.g. dimensions and rigidity).
A number of robots designed to specifically handle SBS microplates have been developed. These robots may be liquid handlers which aspirate or dispense liquid samples from and to these plates, or “plate movers” which transport them between instruments. Also plate readers have been developed, which can detect specific biological, chemical or physical events in samples being processed in the plates.
Adherence to the SBS Microtiter Plate Standards has allowed the easy integration of robotics solutions such as liquid handling machines into the sample preparation process which has had a profound impact on the ability to increase sample throughput. It can therefore be concluded that innovations that will further increase sample throughput must do so without compromising the ability to work within the SBS specifications.
EP-publication 408280 discloses a specially designed multiwell plate format for processing of samples manually in parallel fashion. Publications related to parallel processing of microscope slides and similar plates include WO 99/61152, DE 10002666, US 2004/071605 and US 2005/135974.